EP3202917B1 - Kit zur typisierung zirkulierender tumorzellen - Google Patents

Kit zur typisierung zirkulierender tumorzellen Download PDF

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EP3202917B1
EP3202917B1 EP16734165.0A EP16734165A EP3202917B1 EP 3202917 B1 EP3202917 B1 EP 3202917B1 EP 16734165 A EP16734165 A EP 16734165A EP 3202917 B1 EP3202917 B1 EP 3202917B1
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sequence
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capture probe
gene
dna
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EP3202917A1 (de
EP3202917A4 (de
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Jiasen Xu
Shiyang Wu
Suyan Liu
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Surexam Bio Tech Co Ltd
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Definitions

  • the present disclosure belongs to the field of molecular biology, and relates to the medical sciences and biological technologies. More particularly, it relates to a circulating tumour cell typing and identification kit.
  • Circulating tumour cell refers to collectively all types of tumour cells occurring in peripheral blood, which was detached from a solid tumour lesion (primary lesion or metastatic lesion) spontaneously or due to medical operations. After entering into peripheral blood, most CTCs ended in apoptosis or being devoured, while a few may escape and anchor to develop into a metastatic lesion, resulting in increased mortality risk of malignant tumour patients.
  • tumour cells during the process of entering peripheral blood (PB) circulation, may go through epithelial-mesenchymal transition (EMT).
  • EMT epithelial-mesenchymal transition
  • Some researchers detected the expression of CTC mesenchymal marker vimentin and epithelial marker Keratin mRNAs in metastatic NSCLC patients using immunofluorescent staining, wherein results indicating that strong co-expression of both markers were observed in nearly all CTCs. No CTCs that only expressed keratin mRNA were observed, while a few CTCs that only expressing vimentin mRNA were found in 3 patients.
  • EMT-relevant transcription factor mRNAs TWIST1, SNAIL1, SLUG, ZEB1, FOXC2
  • TWIST1, SNAIL1, SLUG, ZEB1, FOXC2 EMT-relevant transcription factor mRNAs
  • CTCs use epithelial markers (such as epithelial cell adhesion molecule, EPCAM) as target spots, then capture CTCs with corresponding antibodies, and use CKs expressed on cells as a primary diagnostic indicator.
  • epithelial markers such as epithelial cell adhesion molecule, EPCAM
  • CKs expressed on cells as a primary diagnostic indicator.
  • a representative detection method in this regard is CellSearch system, which is the only one that is approved by USFDA for clinical application. False positive or false-negative detection results may occur due to the possible presence of a number of non-neoplastic epithelial cells in peripheral blood, potential contamination in blood samples by normal epithelial cells during blood sampling, and that some CTCs cannot be detected due to loss of epithelial antigens during EMT.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • One goal of the present disclosure is to provide a circulating tumour cell typing and identification kit with strong specificity and high sensitivity.
  • a circulating tumour cell typing and identification kit comprises a capture probe, an amplification probe, and a labeled probe for each marker gene mRNA, wherein the marker gene mRNA comprises the following two types: at least six epithelial cell marker gene mRNAs selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20 mRNAs; and, at least six mesenchymal cell marker gene mRNAs selected from the group consisting of VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1, and SNAI2 mRNAs; wherein, the capture probe binds the marker gene mRNA to the amplification probe and has the following base composition from 5'-terminal to 3'-terminal in sequence: a specific sequence P1 for joining to the marker gene mRNA to be detected, a spacer
  • the marker gene mRNA may further comprise a type for leukocyte marker gene mRNA, wherein the leukocyte marker gene mRNA may be CD45.
  • leukocyte marker gene mRNA may assist further distinguishing leukocytes from tumour cells. Accordingly, interference of leukocytes to detection results is excluded to improve accuracy of results.
  • the P1 specific sequences for the EPCAM gene are SEQ ID NO.1 ⁇ SEQ ID NO.10
  • the P1 specific sequences for the E-cadherin gene are SEQ ID NO.11 ⁇ SEQ ID NO.20
  • the P1 specific sequences for the CEA gene are SEQ ID NO.21 ⁇ SEQ ID NO.30
  • the P1 specific sequences for the KRT5 gene are SEQ ID NO.31 ⁇ SEQ ID NO.40
  • the P1 specific sequences for the KRT7 gene are SEQ ID NO.41 ⁇ SEQ ID NO.50
  • the P1 specific sequences for the KRT17 gene are SEQ ID NO.51 ⁇ SEQ ID NO.60
  • the P1 specific sequences for the KPT20 gene are SEQ ID NO.61 ⁇ SEQ ID NO.70
  • the P2 sequence of the capture probe for the epithelial cell marker genes is SEQ ID NO.181
  • within the amplification probes for the epithelial cell marker gene mRNAs the P2 sequence of the capture probe for the
  • the P1 specific sequences for the VIMENTIN gene are SEQ ID NO.71 ⁇ SEQ ID NO.80
  • the P1 specific sequences for the N-cadherin gene are SEQ ID NO.81 ⁇ SEQ ID NO.90
  • the P1 specific sequences for the TWIST1 gene are SEQ ID NO.91 ⁇ SEQ ID NO.100
  • the P1 specific sequences for the AKT2 gene are SEQ ID NO.101 ⁇ SEQ ID NO.110
  • the P1 specific sequences for the ZEB2 gene are SEQ ID NO.111 ⁇ SEQ ID NO.120
  • the P1 specific sequences for the ZEB1 gene are SEQ ID NO.121 ⁇ SEQ ID NO.130
  • the P1 specific sequences for the FOXC1 gene are SEQ ID NO.131 ⁇ SEQ ID NO.140
  • the P1 specific sequences for the FOXC2 gene are SEQ ID NO.141 ⁇ SEQ ID NO.150
  • the P1 specific sequences for the SNAI1 gene are SEQ ID NO.
  • the P1 specific sequences for the CD45 gene may be two or more selected from SEQ ID NO.171 ⁇ SEQ ID NO.180; the P2 sequence of the capture probe for the leukocyte cell marker gene may be SEQ ID NO.183; and within the amplification probes for the leukocyte cell marker gene mRNA, the P3 sequence may be SEQ ID NO.186 and the P4 sequence may be SEQ ID NO.189.
  • the spacer arm sequence may be 5-10T.
  • the fluorescent group may be selected from the group consisting of: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, and Alexa Fluor 488, and wherein different fluorescent groups are used for marker genes of different cell types.
  • a process for typing and identification of circulating tumour cells mainly comprising the following steps:
  • the step of detecting the presence of epithelial cell marker gene mRNA and/or mesenchymal cell marker gene mRNA comprises following steps:
  • the circulating tumour cell typing and identification kit disclosed in this example can take two forms, either with or without a labeled probe.
  • a capture probe consists of three components from 5'-terminal to 3'-terminal in sequence: a sequence P1 which complementarily pairs with corresponding marker gene mRNA, a spacer arm sequence, and a P2 sequence that is able to complementarily pair with a P3 sequence of a corresponding amplification probe, wherein the same P2 sequence is used in capture probes for marker genes of the same type.
  • the spacer arm separates the P2 sequence of a capture probe from the target mRNA.
  • a spacer arm sequence of appropriate length is usually provided within a probe to reduce steric hindrance and improve hybridization efficiency and specificity.
  • the spacer arm of a capture probe of the present disclosure is preferably 5-10T, and more preferably 5T in this example.
  • Ten capture probes are designed for each marker gene to increase detection specificity.
  • detection for each target gene can be accomplished with two or more selected capture probes with excellent specificity and consistency. Please refer to, for example, Example 8.
  • This example preferably uses 10 capture probes to achieve best specificity.
  • the capture probes for corresponding marker genes are listed in Table 1 and the P2 sequences of the capture probes for different types of marker genes are listed in Table 2.
  • Table 1 P1 sequence of a capture probe for a target gene Gene P1 sequence of capture probe (5' ⁇ 3') SEQ ID NO.
  • An amplification probe is a sequence that links a capture probe and a signal detection component and consists of three components: a P3 sequence at the 5'-terminal that is able to complementarily pair with the P2 sequence of the capture probe, a spacer arm sequence, and a P4 sequence at the 3'-terminal that is able to complementarily pair with the labeled probe (In case that the detection is performed without a labeled probe, the P4 sequence is modified with a fluorescent group at the 3'-terminal. See e.g., the kit B).
  • the spacer arm sequence is formed in the middle with a length of 5 T (The spacer arm of a capture probe of the present disclosure is preferably 5-10 T, and more preferably 5 T in this example).
  • the P3 sequences of the amplification probes for marker genes are listed in Table 3. There is no hairpin structure in the P4 sequence, no dimer or mispairing formed within or among the probes, no specific binding sequences between the P4 sequence and P1, P2, P3, or total mRNA.
  • Preferred base compositions for the P4 sequences in this embodiment are listed in Table 4.
  • Table 3 P3 sequence of an amplification probe for a target gene Type Gene P3 sequence of amplification probe (5' ⁇ 3') SEQ ID NO.
  • a labeled probe consists of two components: a P5 sequence at 5'-terminal that is able to complementarily pair with the P4 sequence of the corresponding capture probe and a fluorescent group at 3'-terminal as a label, so that a target mRNA signal is amplified in cascade via specifically binding to the P4 sequence of the amplification probe.
  • a fluorescent group is not present at the 3'-terminal of the P4 sequence but the 3'-terminal of the labeled probe. See e.g., the kit A).
  • the fluorescent group of the labeled probe may be selected from the group consisting of: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, and Alexa Fluor 488.
  • the fluorescent groups, and the colors or emission wavelengths thereof, selected for FL1, FL2, and FL3 of the labeled probes are different from each other, so as to distinguish marker genes of different types.
  • Preferred fluorescent groups for the labeled probes in this embodiment are shown in Table 6.
  • Table 6 Labeled probe Type Gene P5 sequence of labeled probe SEQ ID NO.
  • the 3'-terminal of the P4 sequence is modified with a fluorescent group.
  • a fluorescent group modification for a P4 sequence of an epithelial cell marker gene detection probe is called FL1
  • a fluorescent group modification for a P4 sequence of a mesenchymal cell marker gene detection probe called FL2
  • a fluorescent group modification for a P4 sequence of an leukocytel marker gene detection probe called FL3.
  • the fluorescent groups may be selected from the group consisting of: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, and Alexa Fluor 488.
  • the fluorescent groups, and the colors or emission wavelengths thereof, selected for FL1, FL2, and FL3 of the labeled probe are different from each other, so as to distinguish marker genes of different types.
  • Preferred 3'-fluorescent groups for the P4 sequences of the marker gene amplification probes are shown in Table 5.
  • Table 5 Dye label of a marker gene Type Marker gene Modified fluorescent group Epithelial cell marker gene EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, KRT20 Cy3 Red fluorescent signal Mesenchymal cell marker gene VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1, SNAI2 Alexa Fluor488 Green fluorescent signal Leukocyte marker gene CD45 Cy5 Purple fluorescent signal
  • Example 2 Detection of circulating tumour cells in peripheral blood of tumour patients using the kit A according to Example 1.
  • solution Formulation Preserving solution 16.6g NH4Cl, 2g KHCO3, 8ml 0.5M EDTA, filled to 1L with ultrapure water Fixing agent 10% Neutral formalin PBS 2.9g Na2HPO4 ⁇ 12H2O, 0.3g NaH2PO4 ⁇ 2H2O, 8.06g NaCl, 0.2g KCI, filled to 1000ml with ultrapure water, pH7.4 Permeabilization agent 0.25% TritonX-100 Digestive enyzme 1ug/ml proteinase K Probe buffer solution 100 ⁇ g/ml denatured salmon sperm DNA, 100 mM LiCI, 0.1% sodium dodecyl sulfate, 9mM EDTA, 50mM HEPES (pH7.5), 0.05% ProClin 300 Amplification buffer solution 6mM Tris-HCl (pH8.0), 1% s odium dodecyl sulfate, 1
  • Each of the probe mixture solution, amplification mixture solution, and color-develpping mixture solution in this example employs all the probes shown in the tables of corresponding genes for the circulating tumour cell typing and identification kit A with a labeled probe according to Example 1.
  • a digestive enzyme treating solution was prepared with corresponding concentration.
  • Reagent composition Dosage per sample Digestive enzyme 1.25 ⁇ l PBS 48.75 ⁇ l Total volume 50 ⁇ l 2.
  • the digestive enzyme working solution was vortex-mixed evenly, dispensed to a 24-well plate at 50 ⁇ l per well.
  • the filter membranes were removed and placed, upturned, on the surface of the digestive enzyme working solution in a 24 well plate, wherein it was ensured that the downward side of the membrane was in sufficient contact with the liquid without bubbles, and stood at room temperature for 1h. 4.
  • the liquid was removed, washed three times with 1ml per well of PBS, each time soaking for 2 min.
  • the filter membranes were kept in PBS buffer until the next experimental operation.
  • Probe hybridization specific probe sequence binding with a target mRNA sequence
  • Probe buffer solution, amplification buffer solution, and color-developing buffer solution were preheated with 40°C water bath for 20min before use.
  • the probe working solution was prepared as follows: Reagent composition Dosage per sample Probe mixture solution 8 ⁇ l Probe mixture solution (preheated at 40°C) 42 ⁇ l Total volume 50.0 ⁇ l vortex-mixed evenly, dispensed to a 24-well plate at 50 ⁇ l per well. 3.
  • the filter membranes were removed and placed, upturned, on the surface of the probe working solution in a 24 well plate, wherein it was ensured that the downward side of the membrane was in sufficient contact with the liquid without bubbles.
  • the liquid was removed, washed three times with 1ml per well of washing solution, each time soaking for 2 min.
  • the filter membranes were kept in the washing solution until the next experimental operation. The soaking time of sample in the washing solution should not exceed 30 min.
  • the amplification working solution was prepared as follows: Reagent composition Dosage per sample Amplification mixture solution 2 ⁇ l Amplification mixture solution (preheated at 40°C) 48 ⁇ l Total volume 50.0 ⁇ l vortex-mixed evenly, dispensed to a 24-well plate at 50 ⁇ l per well. 2. The filter membranes were removed and placed, upturned, on the surface of the amplification working solution in a 24 well plate, wherein it was ensured that the downward side of the membrane was in sufficient contact with the liquid without bubbles. 3. Place the lid on the 24-well plate and incubate for 30 minutes at 40 ⁇ 1°C. 4. The liquid was removed, washed three times with 1ml per well of washing solution, each time soaking for 2 min. The filter membranes were kept in the washing solution until the next experimental operation. The soaking time of sample in the washing solution should not exceed 30 min.
  • the color-developing working solution was prepared as follows: Reagent composition Dosage per sample Color-developing mixture solution 2 ⁇ l Color-developing mixture solution (preheated at 40°C) 48 ⁇ l 50.0 ⁇ l Total volume vortex-mixed evenly in shaded condition, dispensed to a 24-well plate at 50 ⁇ l per well. 2. The filter membranes were removed and placed, upturned, on the surface of the color-developing working in a 24 well plate, wherein it was ensured that the downward side of the membrane was in sufficient contact with the liquid without bubbles. 3. Place the lid on the 24-well plate and incubate for 30 minutes at 40 ⁇ 1°C. 4. The liquid was removed, washed three times with 1ml per well of washing solution, each time soaking for 2 min. The filter membranes were kept in the washing solution until the next experimental operation. The soaking time of sample in the washing solution should not exceed 30 min.
  • DAPI as a nuclear fluorescence group, which emits blue fluorescence signal, was used as the reference substance of the present disclosure.
  • a small amount of circulating tumuor cells and remaining leukocytes were enriched on a filter membrane.
  • the criteria for identifying circulating tumour cells are as follows (see Figure 1 ):
  • This kit utilizes multiple RNA probes specific for multiple CTCs-specific genes.
  • CTCs can be further typed with fluorescent signals of various colors.
  • Type I the epithelial type
  • Type III the mesenchymal type
  • Type II the mixed epithelial-mesenchymal type
  • Type I-specific and Type III-specific genes displaying red and green fluorescent signal spots simultaneously. See Figure 2 .
  • the typing criterias for CTCs are as follows: Table 8 Typing criterias forpositive CTCs Type Cy3 Alexa Fluor 488 Cy5 DAPI I + - - + II + + - + III - + - +
  • Example 3 Detection of cell lines using the kit A according to Example 1
  • the epithelial cell marker genes are two or more selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20;
  • the mesenchymal cell marker genes are two or more selected from the group consisting of VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1, and SNAI2;
  • the leukocyte marker gene is CD45.
  • the epithelial marker genes, mesenchymal marker genes, and leukocyte marker genes provided in this disclosure are genes selected by the inventors through numerous experiments and statistical analysis that specifically express on CTCs, with good specificity and accuracy for identification and typing of CTCs.
  • an epithelial type cell line MCF-10A, a mesenchymal type tumour cell line U118, and a mixed epithelial-mesenchymal type lung cancer cell line PC-9 were used for experiments, while CCRF-HSB-2 lymphocytoblast was used as a negative control.
  • a person skilled in the art will be able to obtain these cell lines commercially as long as the cell line names are provided.
  • About 1000 MCF-10A cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 21-25.
  • About 1000 U118 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 26-30.
  • All the probes in the kit A according to Example 1 including all the marker genes: the epithelial cell marker genes EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20; and the mesenchymal cell marker genes VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1, and SNAI2) and the leukocyte marker gene CD45 were used.
  • Samples 21-40 were tested according to the detection process and method according to Example 2. 50 cells with DAPI blue fluorescent signals in each sample were read, wherein cell numbers in the samples were selected by automatic scanning of a fluorescence microscope.
  • kits disclosed herein can accomplish detection of epithelial, mesenchymal, and leukocyte marker genes with a 100% detection rate for lymphocytoblasts in the cell lines MCF-10A, U118, PC-9, and CCRF-HSB-2 in the samples.
  • Expression of epithelial and/or mesenchymal cell marker genes was only detected in the cell lines MCF-10A, U118, and PC-9, but not in the lymphocytoblasts. Therefore, it indicates that the kit disclosed herein can effectively distinguish tumour cells and leukocytes in a sample.
  • the ephithelial and mesenchymal marker genes selected for use in the kit disclosed herein can accurately type the cells detected (the epithelial type, mesenchymal type, and mixed epithelial-mesenchymal type), by only detecting the fluorescent signals for ephithelial marker genes in the epithelial type cell lines, only detecting the fluorescent signals for mesenchymal marker genes in the mesenchymal type cell lines, and detecting the fluorescent signals for both ephithelial and mesenchymal marker genes in the mixed epithelial-mesenchymal type cell lines. Therefore, the results indicate good specificity and accuracy of the cell-typing markers selected for use in the kit disclosed herein.
  • the epithelial cell marker genes of the kit disclosed herein are selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20, wherein choices were made in accordance with each experimental group; and the mesenchymal cell marker genes are: VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1, and SNAI2.
  • composition, numbers, detection method, and the like, of the capture probes, amplification probes, and labeled probes corresponding to each group of marker genes of this embodiment are described in the kit A of Example 1 as well as in Example 2.
  • Table 11 Selection of epithelial cell marker genes Type Gene types Experimental group Epithelial cell marker gene EPCAM 1 EPCAM, E-cadherin 2 EPCAM, E-cadherin, CEA, KRT5, KRT7 3 EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, KRT20 4
  • mixed epithelial-mesenchymal type liver cancer cell line HepG2, breast cancer cell line MCF-7, and lung cancer cell line PC-9 were used for students.
  • a person skilled in the art will be able to obtain these cell lines commercially as long as the cell line names are provided.
  • About 1000 HepG2 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 41-45.
  • About 1000 MCF-7 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 46-50.
  • About 1000 PC-9 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 51-55.
  • Samples 41-55 were tested using the kit prepared with marker genes from Table 11 and according to the detection process and method according to Example 2.
  • 50 cells with DAPI blue fluorescent signals in each sample were read, wherein cell numbers in the samples were selected by automatic scanning of a fluorescence microscope.
  • the number of fluorescent spots of a corresponding color was read for the 50 cells and an average spot number was calculated.
  • the specific detection results are as follows (Data in Table 12 are cell numbers and data in Table 13 are average numbers of fluorescent spots): Table 13 Comparison of average numbers of fluorescent spots using different numbers of epithelial cell marker genes. No.
  • Comparison of experimental results of Experimental Groups 1-4 demonstrated that, when one, two, five, or seven kinds of epithelial cell marker genes were chosen and all mesenchymal cell marker genes were used, stable detection results for mesenchymal cell marker genes were generated, while variations were seen in detection results for epithelial cell marker genes. Due to the variation in expression of different tumour cell epithelial cell marker genes, detection using only one marker gene results in certain degree of missing detection. Additionally, the detection results stablize when two or more marker genes were used, while the average red fluorescent signal spot gradually increases from Experimental group 1 to 4, as the nubmer of epithelial cell marker gene increases, with improving detection performance. The detection signals were strongest and most stable, with the best performance, when all epithelial cell marker genes were used.
  • Example 5 Detection marker genes selected for a kit.
  • the epithelial cell marker genes of the kit disclosed herein are selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20; and the mesenchymal cell marker genes are: VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1, and SNAI2.
  • Experimental groups 5-8 were designed by respectively choosing two, four, twelve, and seventeen kinds of marker genes for comparison of the detection results thereof.
  • Marker gene CD45 was used for leukocytes. Specific design is shown in Table 14.
  • mixed epithelial-mesenchymal type liver cancer cell line HepG2, breast cancer cell line MCF-7, and lung cancer cell line PC-9 were used for students.
  • a person skilled in the art will be able to obtain these cell lines commercially as long as the cell line names are provided.
  • About 1000 HepG2 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 56-60.
  • About 1000 MCF-7 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 61-65.
  • About 1000 PC-9 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 66-70.
  • Samples 56-70 were tested using the kit prepared as described above and following the detection process and method according to Example 2.
  • 50 cells with DAPI blue fluorescent signals in each sample were read, wherein cell numbers in the samples were selected by automatic scanning of a fluorescence microscope.
  • For fluorescent signal intensity of a target detection marker the number of fluorescent spots of a corresponding color was read for the 50 cells and an average spot number was calculated.
  • the specific detection results are as follows (Data in Table 15 are cell numbers and data in Table 16 are average numbers of fluorescent spots): Table 16 Comparison of average numbers of fluorescent spots using kits with different numbers of marker genes. No.
  • the epithelial cell marker genes (7 genes in total) were used for example, empolying different spacer arms respecitvely.
  • the composition, numbers, detection method, and the like, of the other capture probes, amplification probes, and labeled probes corresponding to each group of marker genes of this embodiment are described in the kit A of Example 1,, except for the only difference in their spacer arms, and the detection methods were described in Example 2.
  • Spacer arms are the same in corresponding capture probes nad amplification probes.
  • Table 17 Spacer arm and length thereof Spacer arm type Length Experimental group poly (dT) 5 9 poly (dA) 8 10 (CH2) n 15 11 poly (TTG) 3 12
  • mixed epithelial-mesenchymal type liver cancer cell line HepG2, breast cancer cell line MCF-7, and lung cancer cell line PC-9 were used for students.
  • a person skilled in the art will be able to obtain these cell lines commercially as long as the cell line names are provided.
  • About 1000 HepG2 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 71-75.
  • About 1000 MCF-7 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 76-80.
  • About 1000 PC-9 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 81-85.
  • Samples 71-85 were tested using the kit prepared with marker genes from Table 11 and following the detection process and method according to Example 2. 50 cells with DAPI blue fluorescent signals in each sample were read, wherein cell numbers in the samples were selected by automatic scanning of a fluorescence microscope. The specific detection results are as follows (Data in the table are cell numbers):
  • signal detection components there are two choices of signal detection components for the kit disclosed herein: 1) a fluorescent group as a 3'-terminal modification in the P4 sequence of the amplification probe; and 2) the P5 sequence of the labeled probe being able to bind to the 3'-terminal P4 sequence of the amplification probe and 3'-terminal fluorescent group being present at the 3' terminal of the labeled probe.
  • Either of the two signal detection components can implement signal amplification and normal signal detection.
  • the use of the labeled probe modified with a fluorescent group allows more stable detection signal and better performance.
  • kit compositions are:
  • mixed epithelial-mesenchymal type liver cancer cell line HepG2, breast cancer cell line MCF-7, and lung cancer cell line PC-9 were used for students.
  • a person skilled in the art will be able to obtain these cell lines commercially as long as the cell line names are provided.
  • About 1000 HepG2 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 86-90.
  • About 1000 MCF-7 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 91-95.
  • About 1000 PC-9 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 96-100.
  • Samples 86-100 were tested using the kit prepared as described above and following the detection process and method according to Example 2. 50 cells with DAPI blue fluorescent signals in each sample were read, wherein cell numbers in the samples were selected by automatic scanning of a fluorescence microscope. For fluorescent signal intensity of a target detection marker, the number of fluorescent spots of a corresponding color was read for the 50 cells and an average spot number was calculated.
  • the specific detection results are as follows (Data in Table 20 are cell numbers and data in Table 21 are average numbers of fluorescent spots): Table 20 Comparison of detection results of epithelial cell marker genes using different signal detection probes. No.
  • the detection results of the two designs were statistically analyzed and demonstrated no variation in the detection results of the cell numbers in the samples bewteen the two experimental designs. Therefore, the two signal detection components are equally effective in their performance.
  • the labeled probes modified with a fluorescent group i.e., Experimental Group 14
  • more epithelial cell marker gene fluorescence spots were detected, the signals were more stable, and the performance thereof was better.
  • the experimental results of detection in mesenchymal cell marker genes are consistent with those of epithelial cell marker genes and the specifc data thereof are not shown.
  • the circulating tumour cell typing and identification kit designed 10 capture probes for each marker gene of different cell types, respecitvely.
  • the same P2 sequence is used for the capture probes for the marker genes of the same cell type.
  • detection for each target gene can be accomplished with desired specificity and consistency by selecting at least two corresponding capture probes.
  • the capture probe number for the epithelial cell marker gene EPCAM was used as an example. See Experimental Groups 15-17, in which 1, 2, and 10 capture probes were chosen, respectively for comparison of the detection results thereof. In this comparative experiment, only EPCAM was used for epithelial cell marker gene, while all genes and probes for the mesenchymal and leukocyte marker genes listed in the kit A according to Example 1 were used.
  • mixed epithelial-mesenchymal type liver cancer cell line HepG2, breast cancer cell line MCF-7, and lung cancer cell line PC-9 were used for students.
  • a person skilled in the art will be able to obtain these cell lines commercially as long as the cell line names are provided.
  • About 1000 HepG2 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 101-105.
  • About 1000 MCF-7 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 106-110.
  • About 1000 PC-9 cells (confirmed with a cytometer) were taken, mixed evenly, and divided into five equal aliquots and numbered as 111-115.
  • Samples 101-115 were tested using the kit prepared as described above and following the detection process and method according to Example 2.
  • 50 cells with DAPI blue fluorescent signals in each sample were read, wherein cell numbers in the samples were selected by automatic scanning of a fluorescence microscope.
  • For fluorescent signal intensity of a target detection marker the number of fluorescent spots of a corresponding color was read for the 50 cells and an average spot number was calculated.
  • the specific detection results are as follows (Data in Table 23 are cell numbers and data in Table 24 are average numbers of fluorescent spots): Table 23 Comparison of detection results for epithelial cell marker gene EPCAM using different numbers of capture probes. No.
  • kits targeted at using different numbers of capture probes for epithelial, mesenchymal, and leukocyte marker genes also had stable and reliable resutls, though the specifc data thereof are not shown.

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Claims (6)

  1. Kit zur Typisierung und Identifizierung zirkulierender Tumorzellen, umfassend eine Fangsonde, eine Amplifikationssonde und eine markierte Sonde für jede Markergen-mRNA, wobei die Markergen-mRNA die folgenden zwei Typen umfasst: wenigstens sechs Epithelzell-Markergen-mRNAs, ausgewählt aus der Gruppe bestehend aus EPCAM-, E-Cadherin-, CEA-, KRT5-, KRT7-, KRT17- und KRT20-mRNAs; und wenigstens sechs Mesenchymzell-Markergen-mRNAs, ausgewählt aus der Gruppe bestehend aus VIMENTIN-, N-Cadherin-, TWIST1-, AKT2-, ZEB2-, ZEB1-, FOXC1-, FOXC2-, SNAI1- und SNAI2-mRNAs; wobei
    die Fangsonde die Markergen-mRNA an die Amplifikationssonde bindet und vom 5'-Ende zum 3'-Ende der Reihe nach die folgende Basenzusammensetzung aufweist: eine spezifische Sequenz P1 zum Verbinden mit der nachzuweisenden Markergen-mRNA, eine Spacer-Arm-Sequenz und eine P2-Sequenz, wobei es in der P2-Sequenz keine Haarnadelstruktur gibt, kein Dimer oder keine Fehlpaarung, das bzw. die innerhalb oder zwischen den Sonden gebildet wird, keine spezifischen Bindungssequenzen zwischen der P2-Sequenz und P1, P4, oder der Markergen-mRNA, und wobei dieselbe P2-Sequenz für die Fangsonden für die Markergene desselben Typs verwendet wird;
    die Amplifikationssonde vom 5'-Ende zum 3'-Ende der Reihe nach die folgende Basenzusammensetzung aufweist: eine P3-Sequenz, die in der Lage ist, sich mit der P2-Sequenz der zugehörigen Fangsonde komplementär zu paaren, eine Spacer-Arm-Sequenz und eine P4-Sequenz, wobei es keine Haarnadelstruktur in der P4-Sequenz gibt, kein Dimer oder keine Fehlpaarung, das bzw. die innerhalb oder zwischen den Sonden gebildet wird, keine spezifischen Bindungssequenzen zwischen der P4-Sequenz und P1, P2, P3, oder der gesamten mRNA; und
    jede markierte Sonde eine P5-Sequenz aufweist, die in der Lage ist, sich mit der P4-Sequenz der zugehörigen Amplifikationssonde komplementär zu paaren, und eine fluoreszierende Gruppe als terminale Modifikation, wobei verschiedene fluoreszierende Gruppen für Markergene verschiedener Zelltypen verwendet werden;
    wobei, innerhalb der Fangsonde für das Epithelzell-Markergen, die P1-spezifischen Sequenzen für das EPCAM-Gen SEQ ID NO.1 - SEQ ID NO.10 sind, die P1-spezifischen Sequenzen für das E-Cadherin-Gen SEQ ID NO.11 - SEQ ID NO.20 sind, die P1-spezifischen Sequenzen für das CEA-Gen SEQ ID NO.21 - SEQ ID NO.30 sind, die P1-spezifischen Sequenzen für das KRT5-Gen SEQ ID NO.31 - SEQ ID NO.40 sind, die P1-spezifischen Sequenzen für das KRT7-Gen SEQ ID NO.41 - SEQ ID NO.50 sind, die P1-spezifischen Sequenzen für das KRT17-Gen SEQ ID NO.51 - SEQ ID NO.60 sind und die P1-spezifischen Sequenzen für das KRT20-Gen SEQ ID NO.61 - SEQ ID NO.70 sind; die P2-Sequenz der Fangsonde für die Epithelzell-Markergene SEQ ID NO.181 ist; und innerhalb der Amplifikationssonden für die Epithelzell-Markergen-mRNAs die P3-Sequenz SEQ ID NO.184 ist und die P4-Sequenz SEQ ID NO.187 ist; und
    wobei, innerhalb der Fangsonde für das Mesenchymzell-Markergen, die P1-spezifischen Sequenzen für das VIMENTIN-Gen SEQ ID NO.71 - SEQ ID NO.80 sind, die P1-spezifischen Sequenzen für das N-Cadherin-Gen SEQ ID NO.81 - SEQ ID NO.90 sind, die P1-spezifischen Sequenzen für das TWIST1-Gen SEQ ID NO.91 - SEQ ID NO.100 sind, die P1-spezifischen Sequenzen für das AKT2-Gen SEQ ID NO.101 - SEQ ID NO.110 sind, die P1-spezifischen Sequenzen für das ZEB2-Gen SEQ ID NO.111 - SEQ ID NO.120 sind, die P1-spezifischen Sequenzen für das ZEB1-Gen SEQ ID NO.121 - SEQ ID NO.130 sind, die P1-spezifischen Sequenzen für das FOXC1-Gen SEQ ID NO.131 - SEQ ID NO.140 sind, die P1-spezifischen Sequenzen für das FOXC2-Gen SEQ ID NO.141 - SEQ ID NO.150 sind, die P1-spezifischen Sequenzen für das SNAI1-Gen SEQ ID NO.151 - SEQ ID NO.160 sind, die P1-spezifischen Sequenzen für das SNAI2-Gen SEQ ID NO.161 - SEQ ID NO.170 sind; die P2-Sequenz der Fangsonde für die Mesenchymzell-Markergene SEQ ID NO.182 ist; und innerhalb der Amplifikationssonden für die Mesenchymzell-Markergen-mRNAs die P3-Sequenz SEQ ID NO.185 ist und die P4-Sequenz SEQ ID NO.188 ist.
  2. Kit zur Typisierung und Identifizierung zirkulierender Tumorzellen nach Anspruch 1, wobei die Markergen-mRNA ferner einen Typ für Leukozyten-Markergen-mRNA umfasst, wobei die Leukozyten-Markergen-mRNA CD45 ist.
  3. Kit zur Typisierung und Identifizierung zirkulierender Tumorzellen nach einem der Ansprüche 1 - 2, wobei innerhalb der Fangsonde für die Leukozytenzell-Markergen-mRNA die P1-spezifischen Sequenzen für das CD45-Gen zwei oder mehr ausgewählt aus SEQ ID NO.171 - SEQ ID NO.180 sind; die P2-Sequenz der Fangsonde für die Leukozytenzell-Markergene SEQ ID NO.183 ist; und innerhalb der Amplifikationssonden für die Leukozytenzell-Markergen-mRNA die P3-Sequenz SEQ ID NO.186 ist und die P4-Sequenz SEQ ID NO.189 ist.
  4. Kit zur Typisierung und Identifizierung zirkulierender Tumorzellen nach einem der Ansprüche 1 - 2, wobei die Markergen-mRNAs Folgendes umfassen: die Epithelzell-Markergen-mRNAs von EPCAM, E-Cadherin, CEA, KRT5, KRT7, KRT17 und KRT20; die Mesenchymzell-Markergen-mRNAs von VIMENTIN, N-Cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1 und SNAI2; sowie die Leukozyten-Markergen-mRNA von CD45.
  5. Kit zur Typisierung und Identifizierung zirkulierender Tumorzellen nach einem der Ansprüche 1 - 2, wobei die Spacer-Arm-Sequenz 5-10-Thymin ist.
  6. Kit zur Typisierung und Identifizierung zirkulierender Tumorzellen nach einem der Ansprüche 1 - 2, wobei die fluoreszierende Gruppe aus der Gruppe ausgewählt ist, die aus Folgendem besteht: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 und Alexa Fluor 488, und wobei verschiedene fluoreszierende Gruppen für Markergene verschiedener Zelltypen verwendet werden.
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